Investigation of Relationship Between Chemical Stress Factors and Certain Metabolites Including Cardenolides in Callus Cultures of Endemic Turkish Digitalis L. Species

The aim of the present research is to obtain relationship between different stress treatments [Cu (copper) and Hg (mercury)] and content of cardiac glycosides (digoxigenin, gitoxigenin, lanatoside C, digoxin and digitoxin) as secondary metabolites of commercial value for the pharmaceutical industry and to determine the antioxidant metabolites against stress conditions in callus cultures of endemic Turkish Digitalis species. The effects of different stress treatments on cardiotonic glycoside accumulation in D. lamarckii Ivanina, D. trojana Ivanina, D. davisiana Heywood and D. cariensis Boiss. ex Jaub. et Spach were investigated using HPLC. HPLC analysis revealed that all stress conditions were significantly effective at 5% significance level according to their control groups. The predominant cardiac glycoside was lanatoside C (Lan C) followed by digitoxin, digoxigenin, gitoxigenin and digoxin. No digoxin was detected in all treatments as well as in control groups. For the calibration curves, concentrations of 5, 10, 20, 30 and 40 mg/l digoxigenin, gitoxigenin, lanatoside C, digoxin and digitoxin were used (R= 0.99). Cardenolides were eluted with acetonitrile (A) and water (B) gradients as follows: 0 to 20 min 20% (A), 80% (B); 20 to 23.40 min 30% (A), 70% (B); 23.40 to 30 min 25% (A), 75% (B) and 30 to 40 min 40% (A), 60%(B). Average peak area of the glycoside in samples was automatically calculated and monitored by ChemStation LC/MS software against that of standards. Enhanced production of cardenolides was achieved from callus cultures elicited with 50 μm CuSO4 and HgCl2. Higher amounts of cardenolides were obtained when callus of four Digitalis species were elicited with CuSO4. Results demonstrated that catalase (CAT, EC 1.11.1.6), superoxide dismutase (SOD, EC 1.15.1.1) activities, the total contents of phenolics and proline were markedly stimulated under stress conditions. All these results indicated that treatments have induced changes in the redox state of callus cells and suggest that this alteration change cardenolides accumulation and antioxidative status in Digitalis L. callus cultures.


INTRODUCTION
Digitalis L. produces various cardiac glycosides which have potential to treat many diseases such as edema, myocardial infarction, arterial hypertension, cardiac dysfunction, angina and hyertropy [1].Besides their cardiotonic effects, these compounds are also effective chemotherapeutic agents, especially in breast and prostate cancer treatments [2].Digitalis species is distributed in the Mediterranean region, Western Asia and Europe.Digitalis davisiana Heywood, Digitalis lamarckii Ivanina, Digitalis cariensis Boiss.ex Jaub.et Spach and Digitalis trojana Ivanina are endemic to Turkey [3].Traditionally, cell and organ cultures have been used for production of secondary metabolites, but the yield of cardiac glycosides have been low.Attempts have been made to increase concentration of metabolites in shorter period of time.Exogenous addition of biotic and abiotic elicitors is considered to be one of the most promising strategies for the induction of secondary metabolites production [4].Zhao et al. [5] proposed that these elicitor molecules act as extracellular or intracellular signals and initiate a signal transduction network that is required for the activation of transcription factors, which organize the expression of genes included in plant secondary metabolism [6].Among the various abiotic elicitors, heavy metal stresses have been considered as effective elicitors for the increased production of secondary metabolites in in vitro cultures.Heavy metal stress has become a headmost environmental threat to crop production.Being a potential hazardous factor, toxic metals decrease the plant growth, yield and sustainability of production, thus can cause the alarming situation for food availability.Plants under the stress environment facing the alterations of cellular protein functions, lipid and thylakoid structures.Disturbance or breakage of these structures is directly linked with plant photosystem that can affect the senescence process [7,8].Copper (Cu), microelement, has important physiological functions in plants.At higher concentrations, it leads to physiological and morphological disturbances, as a consequence decrease the yield [9]. Mercury (Hg) is a toxic metal that can be absorbed from the atmosphere and soil.It can be accumulate in the plant organs and cause to phytotoxic effects.The toxic metal threshold level in the tissue is defined by the 'stress point'.Beyond the stress point, the cell will be irreversibly damaged [10].These metabolic changes can directly trigger the plant defense system, including enzymatic and non-enzymatic antioxidants to cope with overproduce of reactive oxygen species (ROS) in the cell [11].As a consequence of unfavorable conditions, activation of genes related to the enzymatic defense system, including catalase (CAT), superoxide dismutase (SOD), guaicol peroxidase (POD), glutathione reductase (GR) and the non-enzymatic antioxidants such as glutathione, ascorbate (vitamin C), carotenoids, αtocopherol (vitamin E), proline and various phenylpropanoid derivatives (phenolic compounds) were observed [12].On the basis of background information, the aim of this work is to investigate the induction of cardenolide compounds (lanatoside C, digitoxin, digoxigenin, gitoxigenin and digoxin) in D. davisiana Heywood, D. lamarckii Ivanina, D. trojana Ivanina and D. cariensis Boiss.ex Jaub.et Spach, in response to application of heavy metal toxicity and to determine the correlation between the cardiotonic glycosides accumulation and stress responsive antioxidant defense system.

Experimental Design
The seeds were cultured on MS medium including containing 3% sucrose and 0.8% agar (pH 5.7-5.8)for germination.Hypocotyl explants were cultured on MS medium including vitamins (0.5 ppm TDZ and 0.25 ppm IAA) (Sahin et al., 2013) for callus induction.After 30 days of culture 50 µm HgCl2 and 50 µm CuSO4 were used in cultures in order to expose the callus cultures to chemical stress for 10 days [13].

Extraction and HPLC analysis of cardenolides
Cardenolide extraction was determined according to the modified method of Wiegrebe and Witchtl [14].Qualitative and quantitative analysis of cardenolides were detected and calculated as previously described HPLC protocol [15].

Total phenolic assay
The total phenolic content were determined using the Folin-Ciocalteau [19] method.The total phenolic content of extracts was expressed as mg gallic acid equivalents (GAE)/ g dw.

Proline analysis
Proline was determined according to the method of Bates et al.,[20].Proline content was calculated as µmol proline /g dw.

Statiscal Analysis
Data were statistically analyzed using SPSS Version 15.0 (SPSS Inc., Chicago, IL, USA) and Duncan's multiple range test at P≤0.05.Each treatment was made in triplicate.

RESULTS
Callus was initiated from hypocotyl explants cultured on MS medium supplemented with 0.25 mg L -1 IAA and 0.5 mg L -1 TDZ.After 30 days, callus was transferred to MS medium containing 50 µm HgCl2 and 50 µm CuSO4 for 10 days.Here, we examined the effects of HgCl2 and CuSO4 as an heavy metal stress factors on cultures which induced cardiotonic glycoside accumulation and antioxidant activities in four Digitalis species (D. cariensis, D. davisiana, D. trojana, D. lamarckii).Application of HgCl2 and CuSO4 to the medium were not affected cell viability.However, the colour of the callus changed from green (control group) to brownish with HgCl2 and CuSO4 applications (Figure 1).Results related to cardenolides accumulation of treated and untreated plants of four Digitalis species (D. davisiana Heywood, D. lamarckii Ivanina, D. trojana Ivanina and D. cariensis Boiss.ex Jaub.et Spach) are shown in Table 1.Addition of HgCl2 and CuSO4 (50 µm) into media significantly affected the cardenolides accumulation as compared to control.Especially, CuSO4 treatments played a pivotal role to the accumulation of cardenolides.Lanatoside C was the predominant cardiac glycoside followed by digoxigenin, digitoxin, gitoxigenin and digoxin.On the other hand, the content of digoxin was below the limit of detection in all treatments.The use of CuSO4 and HgCl2 led to a drastic increase in the accumulation of Lan C in all Digitalis species.The control (non-treated) callus produced 302.04±7.49µg/g dw Lan C while those treated with chemical stress by CuSO4 and HgCl2 producing 721.80±8.78µg/g dw and 377.23±5.20 µg/g dw Lan C respectively in D. lamarckii callus cultures.Similar to D. lamarckii, the positive correlation between the accumulation of Lan C and CuSO4 -HgCl2 applications was detected in D. trojana callus cultures.285.63±2.57µg/g dw Lan C was found in the control (non-treated) callus while those treated with chemical stress by CuSO4 and HgCl2 producing 619.83±7.23 µg/g dw and 346.37±9.47µg/g dw Lan C, respectively.In D. davisiana cultures, the accumulation of Lan C was significantly induced as a concequence of CuSO4 and HgCl2.Lan C of control was 259.85±8.17µg/g dw while those treated with chemical stress by CuSO4 and HgCl2 producing 844.097±7.89µg/g dw and 461.13±6.08 µg/g dw Lan C, respectively.CuSO4 as well as HgCl2 stress was followed by a significantly enhanced accumulation of Lan C in D. cariensis callus cultures.The control (non-treated) callus produced 280.71±7.14µg/g dw Lan C while those cultured on under chemical stress by CuSO4 and HgCl2 producing 939.21±9.09µg/g dw and 673.23±6.58µg/g dw Lan C respectively.Although a noticeable increase was observed in digitoxin, digoxigenin content under the exposure of CuSO4, there was not any significant increase in gitoxigenin, digitoxin, digoxigenin content under the exposure of HgCl2 compared to non-treated callus in D. lamarckii.In D. trojana cultures, digoxigenin (17.13±1.31µg/g dw) and gitoxigenin levels (7.62±1.13µg/g dw) were significantly increased under CuSO4 stress.But it is apparent that, at 5% significance level, there was not any significant change observed between control groups and digitoxin content under both applied treatments.Addition of CuSO4 and HgCl2 into the medium significantly increased the digoxigenin, gitoxigenin and digitoxin content in D. davisiana and D. cariensis callus cultures as compared with respective controls.The data regarding the antioxidant and non-enzymatic antioxidant enzymes are presented in Table 2. Activities of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT) were significantly enhanced in all species under 50 μm CuSO4 and HgCl2 treatments as compared with respective controls.The CAT activity in control group ranged from 8.24 μmol H2O2/min/mg protein for D. cariensis to 19.30 μmol H2O2/min/mg protein for D. lamarckii followed by 15.55 μmol H2O2/min/mg protein for D. trojana.When incubating callus with 50 μm CuSO4 induced CAT activity ranged from 12.66 μmol H2O2/min/mg protein for D. cariensis to 28.61 μmol H2O2/min/mg protein for D. lamarckii followed by 23.50 μmol H2O2/min/mg protein for D. trojana.Addition of 50 μm HgCl2 to the media also enhanced CAT activity ranged from 10.69 μmol H2O2/min/mg protein for D. cariensis to 28.61 μmol H2O2/min/mg protein for D. lamarckii.Exposure of heavy metals was followed by a remarkable increase in SOD activity as compared to the control groups.In control, SOD activity ranged from 0.14 U/mg protein for D. lamarckii to 0.48 U/mg protein for D. davisiana followed by 0.46 U/mg protein for D. cariensis.While that cultured expose with CuSO4 induced SOD activity ranged from 0.55 U/mg protein for D.lamarckii to 1.14 U/mg protein for D. cariensis.Exogenously applied HgCl2 induced SOD activity ranged from 0.31 U/mg protein for D.lamarckii to 0.78 U/mg protein for D. davisiana.Besides the enzymatic defense machinery, we also studied non-enzymatic antioxidant response under different treatments of heavy metals.Non-enzymatic antioxidant named as total phenolic and proline levels were increased in all species under CuSO4 and HgCl2 stress as compared to control.Maximum total phenolic and proline production were determined as 476.95 µg GA mg -1 (1.88 -fold higher than control) and 1.82 µmol g -1 (2.24-fold higher than control) with the elicitation of CuSO4 respectively.

DISCUSSION
Plant secondary metabolism is the source for many fine chemicals of commercial importance.One group of natural products of major interest in the pharmaceutical industry is cardiac glycosides from Digitalis species.Levels of plant carbon-based secondary compounds are partly under genetic control and determined in part by environmental conditions [21] therefore, in order to maximise the production of a specific natural product, it will be necessary to understand the various factors that control and influence its biosynthesis.In the case of Digitalis plants, previous studies have reported that cardenolide biosynthesis is basically dependent on morphological differentiation [22] and genotype [23], although numerous environmental factors may determine, in a greater or lesser degree, plant productivity.Thus, it is known the influence that mineral nutrients [24,25], CO2 and water stress [26], and light conditions [27] exert on cardenolide accumulation.Moreover, in our previous papers, we showed that H2O2 pre-treatment [15] and elimination of Ca, Mg or both from the medium [28] resulted in an increase in cardenolides, enzymatic and non-enzymatic antioxidants in callus cultures of Digitalis davisiana Heywood, Digitalis lamarckii Ivanina, Digitalis trojana Ivanina and Digitalis cariensis Boiss.ex Jaub.et Spach.Elicitation strategies have been widely used to induce the production of secondary metabolites in in vitro cell cultures [29].In the light of earlier studies, it was concluded that H2O2 increase occurred after Cu, Cd [30] and Hg [13] treatment of A. thaliana and S. lycopersicum, respectively.Similarly, Smith et al. [31] showed that production of umbelliferone, which is a phytoalexin produced in response to stress or infection in whole plants, was stimulated in suspension cultures of Ipomoea batatas (L.) Poir.using HgCl2.Korsangruang et al. [32] found that CuSO4 enhanced the accumulation of isoflavonoid compounds in Pueraria candollei cell suspension culture.However, there are not any reports of Digitalis tissue cultures in which improvements in cardenolide production have been achieved by heavy metal treatments.
The more recent identification and characterization of several enzymes/genes involved in pregnane and cardenolide metabolism, such as 3β-hydroxysteroid dehydrogenase and progesterone 5β-reductase.P5βR is considered to be a key enzyme in cardenolide biosynthesis as: it is the first stereospecific enzyme of the pathway leading to 5β-configured derivatives; it appears to be the initial step in cardenolide biosynthesis.indicated that P5βR2 is a critical component for the chemical defense of foxglove plants against herbivores, through cardenolide accumulation, in association with ethylene and H2O2 signaling in Digitalis purpurea.Available information suggests that H2O2 directly regulates the expression of numerous genes involved in plant defense and the related pathways such as antioxidant enzymes, defense proteins and transcription factors [34].In our studies, heavy metal stress significantly induced cardenolide production in callus cultures of all Digitalis species.The increase in secondary metabolite concentrations in the callus cultures under heavy metal stress may also be associated with the alterations in the activity of P5βR2 gene related to cardenolides.This was probably due to the reason that heavy metal-induced ROS generation was responsible for transcriptional activation of genes encoding enzymes involved in cardenolide biosynthesis.Although, the applications of heavy metals significantly increased the cardenolides accumulation in all Digitalis species, there was no digoxin detected in all treatments.It is well known that digilanidase enzyme catalyzed deglucosylation and subsequent deacetylation of Lan C to make into digoxin in the leaves [35].This was probably due to the reason that the amount of digoxin in the callus tissues examined was found to be below the detection limit of the determination used.
In current work, CAT and SOD activity were significantly increased with the CuSO4 and HgCl2.According to , many heavy metals could result in increased activity of NADPH oxidase partially related with O2 -formation.Correspondingly, heavy metalinduced O2 -formation could cause to transcriptional activation of genes responsible for antioxidative enzymes.O2 -formation could be associated with an increased activity of SOD for conversion and parallel increased activity of CAT.Furthermore, Mittler [37] reported that increased level of antioxidants has a pivotal role in deteriorating the ROS activity, thus plants could be able to maintain their physiological functions under the stress environment.Along with primary defense mechanism, plants also activated their non-enzymatic antioxidant system named as phenolics and proline as a result of biotic and abiotic stresses, including heavy metal toxicity [38].Stress induced proline accumulation can reduce photochemical activity losses and the production of free radicals in the thylakoid membrane of the chloroplast [39].Thus, proline contributed to arrest photo inhibitory damage in plants.Many plants accumulate proline at higher concentration in response to toxic concentrations of heavy metals [40].Some researchers conclude that proline accumulation is not related to protection against metal stress [41], is just a symptom of injury.On the contrary, it has been suggested that proline might have an adaptive role related to survival of plants against heavy metal toxicity [42].Zengin and Munzuroglu [43] showed that copper and mercury toxicity increased the proline content of the leaves of bean (Phaseolus vulgaris L.) seedlings.A similar observation was also recorded in our experiment that there were a positive correlation between metal toxicity and proline accumulation in callus cultures of Digitalis species.Therefore, we may conclude that proline may have a protective an adaptive role against stress conditions.
Studies suggested that H2O2 contents increased under both biotic and abiotic stresses induced expression of phenyl ammonia lyase (PAL) accompanied with the de novo synthesis of phenolics [44].Phenolic compounds have antioxidant action because they are particularly bind iron and copper owing to their high tendency to chelate metals [45].The content of free phenols was found to increase in two lines of wheat (T.aestivum) and root cultures of Lupinus albus L. with increasing Cu and Hg concentration in the medium, respectively.[46,47].In our studies, the increase in phenolic levels observed in the callus cultures of Digitalis species to reduce the oxidative stress caused by Cu and Hg.

CONCLUSION
This study demonstrated the role of heavy metals in the stimulated production of cardenolides in the callus cultures of Digitalis species.The productivity of cardenolides was found to be dependent on types of species and two elicitors.The present study has established that CuSO4 is a better elicitor than HgCl2 for cardenolide production from Digitalis callus cultures.This study would help to intentionally manipulate elicitation strategy to improve the yield of cardenolides profile in the callus cultures as well as to extend this protocol for large scale production of cardenolides in bioreactor utilizing Digitalis species in order to cope up with the demand for cardenolides in international pharmaceutical markets in future.Our results also showed that enzymatic and non-enzymatic antioxidative system are sensitive to these stress factors.Different accumulation trends were detected between individual compounds against applied treatments.We expect that further analysis of cardenolides and antioxidant molecules will provide insights into the regulatory relationships among these molecules and the role of these molecules in the establishment of mechanism for cardenolide production.The results presented in this work regarding the analysis of cardenolides under stress conditions are believed to create a useful base for the future studies in understanding the antioxidant mechanism which can be employed for the improvement of a large scale production of cardenolides.

Table 1 .
Results of cardenolides accumulation in the control and elicited callus tissues of Digitalis species.Means followed by different letters in the same column are significantly different (P < 0.05).
Note: LOD; limit of detection.

Table 2 .
Effect of different treatments of heavy metals on enzymatic and non-enzymatic antioxidant activities in the callus cultures of Digitalis species.
Note: Values followed by same small letters are not significantly different at P<0.05.